Exploiting Substrate Promiscuity to Develop Activity-Based Probes for TET Family Enzymes

• Published in: Journal of the American Chemical Society Vol. 140; no. 50; pp. 17329 - 17332
• Main Authors: Ghanty, Uday, DeNizio, Jamie E., Liu, Monica Yun, Kohli, Rahul M.
• Format: Journal Article
• Language: English
• Published: 11.12.2018
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/jacs.8b04722

TET enzymes catalyze repeated oxidations of 5-methylcytosine in genomic DNA. Due to the challenges of track-ing reactivity within a complex DNA substrate, chemical tools to probe TET activity are limited, despite these enzyme’s crucial role in epigenetic regulation. Here, building on precedents from related Fe(II)/α-ketoglutarate-dependent dioxygenases, we show that TET enzymes can promiscuously act upon cytosine bases with unnatural 5-position modifications. Oxidation of 5-vinylcytosine (vC) in DNA results in the predominant formation of a 5-formylmethylcytosine product that can be efficiently labeled to provide an end-point read-out for TET activity. The reaction with 5-ethynylcytosine (eyC), moreover, results in the formation of a high-energy ketene intermediate that can selectively trap any active TET isoform as a covalent enzyme-DNA complex, even in the complex milieu of a total cell lysate. Exploiting substrate promiscuity therefore offers a new and needed means to directly track TET activity in vitro or in vivo .